ABSTRACT This proposal aims to define a new mechanism of antiviral activity by an Interferon stimulated gene (ISG) Oligoadenylate Synthetase 1 (OAS1). The host innate immune response is initiated by the sensing of non-self viral nucleic acid, and largely mediated through type I and III interferons (IFN). IFNs induce expression of hundreds of ISGs, many of which inhibit virus replication in infected cells, protect uninfected neighboring cells, and shape the adaptive immune response to clear virus infection. OASs are a family of ISG that belongs to an evolutionarily ancient family of nucleotidyl-transferases (NTase). The canonical antiviral mechanism of OAS proteins involves the enzymatic synthesis of 2'-5'-oligoadenylates, causing downstream activation of RNase L and leading to the inhibition of protein synthesis. However, the mechanisms of antiviral activity of multiple enzymatically active and inactive OAS isoforms present in human and mouse genomes are not yet clear. We have found that a specific OAS1 isoform enhances the translation of multiple antiviral proteins, including cGAS and IRF1. This OAS1 isoform (OAS1 P46) enhances translation independent of its enzymatic activity and RNase L. Our preliminary results suggest that OAS1 P46 enhances the translation of specific proteins through binding respective mRNAs. In humans, OAS1 P46 is generated due to an alternative splicing event at the C- terminal of the OAS1 gene. A naturally occurring polymorphism (rs10774671, A/G) at this alternative splice site regulates P46 expression, and has been associated with disease severity to multiple virus infections. Using primary human hematopoietic cells, we demonstrate that rs10774671 G allele coding for P46 also enhances IRF1 protein expression in T cells. We also provide multiple evidences suggesting the functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no NTase activity), which similarly affects WNV susceptibility in vivo. Based on these observations, we hypothesize that specific isoforms of OAS1 modulate innate immune responses against viruses through unique NTase activity-independent mechanisms. The goal of this proposal is to determine how OAS1 enhances specific protein translation, and the consequences of this newly identified function of OAS1 on the antiviral innate and adaptive immunity. Our three independent Aims are to: (1) Determine the molecular mechanism of OAS1-mediated translational regulation through biochemical and cell biology approaches; (2) Define the cellular targets mediating the antiviral effect of OAS1 using several gene-deficient cells; and (3) Define the in vivo role of Oas1b during virus infection using a newly generated Oas1b knock-in mouse. Upon completion of this study, we should establish a new paradigm of antiviral activity by OAS proteins that may lead to therapeutic strategies modulating this OAS1-cGAS-type I IFN axis.